5 Rates and energetics of translation At 37 C, the rate of translation in E. coli is about 15 amino acids per second. The translational rate is equivalent to the transcriptional rate which is ~45 nucleotides per second. Energy cost for synthesis of a protein with N amino acids: 2N ATPs to charge trna (ATP -> AMP + PP -> AMP + 2P i ) 1 GTP for initiation (IF2) N-1 GTPs to position trna for N-1 peptide bonds (EF-Tu) N-1 GTPs for N-1 translocation steps (EF-G) 1 GTP for termination (RF-3) ==== 4N Total of 4 high-energy phosphate bonds cleaved per amino acid Each ATP or GTP cleavage generates ~40 kj/mol Each peptide bond costs ~160 kj/mol in the cell, yet an uncatalyzed chemical reaction to form a peptide bond costs only ~20 kj/mol. (1 of 7)5/25/2007 9:39:47 AM

6 Why is it so costly to make a peptide bond on a ribosome? The excess energy is used for generating an accurate, defined polypeptide sequence, not a random one or a combination of multiple possibilities. Two sources of errors during translation: Attachment of an incorrect amino acid to a trna Mispairing of the trna anticodon with the mrna codon Two proofreading mechanisms exist to prevent these errors: Proofreading before aminoacyl adenylate intermediate is attached to trna. Kinetic proofreading before peptide bond formation: A delay is introduced between the binding of an aminoacyl-trna to the codon and the formation of the peptide bond to allow errors to be corrected: EF-Tu-GTP binds an aminoacyl-trna and bring it into the A-site. EF-Tu allows the anticodon to interact with the codon but prevents peptide bond formation. An incorrect trna will bind weakly to the codon and will dissociate from the codon before an incorrect amino acid is incorporated into the polypeptide. Correct codon-anticodon matching triggers hydrolysis of GTP by the EF-Tu, after which EF-Tu-GDP dissociates. Peptide bond formation proceeds. (2 of 7)5/25/2007 9:39:47 AM

7 Each major step in protein synthesis, except peptide-bond formation itself, involves hydrolysis of GTP to GDP. Regulation of protein synthesis PROKARYOTES Short-lived mrna (few minutes), so little need for complicated translational regulation. In prokaryotes, most of the regulation is at the transcriptional level. Rates vary only by a factor of 100. Variance is due to differences in Shine- Dalgarno sequences and how strongly a particular sequence base-pairs with the 16S rrna of the 30S ribosomal subunit. EUKARYOTES Long-lived mrna (hours to days) and thus a greater need to regulate the rate of protein synthesis. (3 of 7)5/25/2007 9:39:47 AM

8 Several known mechanisms: mrna masking: mrna is bound to a variety of proteins that prevent association with ribosomes. When appropriate signal is received, the proteins dissociate from mrna, leaving the transcript free to associate with the ribosome. The signal is usually in the form of phosphorylation/ dephosphorylation. mrna masking is a major form of regulation in early embryonic development. antisense RNA: short segment of RNA, complementary to mrna, that forms double stranded RNA which cannot be translated by ribosome. Two known examples: blockage of protein synthesis of fruit-ripening enzyme in tomatoes the c-myb gene product which promotes smooth muscle development and blockage in injured arteries. Heme Control of Globin Synthesis: Red blood cells are programmed to synthesize large amounts of globin. The globin chains, subsequent to translation, are assembled with heme into hemoglobin. If there is an insufficient supply of heme to insert into the newly synthesized globin chains, then translation is turned off. The lack of heme triggers the accumulation of a heme-controlled inhibitor (HCI) protein. This protein is a kinase which phosphorylates eif2-gtp. The phosphorylation blocks the dissociation of eif2 and eif2-beta that normally occurs in the initiation cycle. Thus, the cell becomes rapidly depleted of unphosphorylated eif2 which is normally recycled for initiation of additonal mrna. Either the (4 of 7)5/25/2007 9:39:47 AM

9 addition of heme, which represses the production of HCI, or the addition of lots of unphosphorylated eif2, which bypasses the HCI effect, can restart initiation again. Interferon: Interferons are glycoproteins that are secreted by virusinfected cells. Interferons prevent additional infection by other types of viruses by inhibiting protein synthesis in infected cells. Two mechanisms of action: induces production of protein kinase, DAI (double-stranded RNAactivated inhibitor) which in the presence of dsrna, phosphorylates eif2-alpha and stabilizes the eif2-alpha-eif2-beta complex in a manner similar to the heme-controlled inhibitor (HCI). induces a cascade effect which ultimately activates an endonuclease, RNase L, that rapidly degrades mrna. Inhibition of protein synthesis by antibiotics (5 of 7)5/25/2007 9:39:47 AM

10 Antibiotics are bacterially or fungally produced substances that inhibit the growth of other organisms. Antibiotics target a wide spectrum of vital processes: they block DNA replication, transcription and bacterial cell wall synthesis. A large number of antibiotics, including medically useful substances, block protein translation. Blocking protein translation is very effective for two reasons: Protein translation plays a central role in overall metabolism The structural differences between prokaryotic and eukaroytic ribosomes and associated factors (IFs/EFs/RFs) allow specific targeting. (6 of 7)5/25/2007 9:39:47 AM

12 Summary (6.3) Summary GTP-binding often causes major conformational changes in proteins Each major step in protein synthesis, except peptide-bond formation itself, involves hydrolysis of GTP to GDP. Four high-energy phosphate-bonds are required for each amino acid added The rate of protein synthesis is well matched to the rate of transcription EF-G is shaped like EF-Tu-GTP-aminoacyl-tRNA (molecular mimicry) mrna in prokaryotes is short-lived (minutes) mrna in eukaryotes is long-lived (hours/days), requiring additional control A large number of antibiotics inhibit protein synthesis, many specifically in prokaryotes Next lecture: Post-Translational Processing Please report typos, errors etc. by (mention the title of this page). 9:39:47 AM

Elongation & Termination of Protein Synthesis (5.1) Lecture 5 1. INITIATION Assembly of active ribosome by placing the first mrna codon (AUG or START codon) near the P site and pairing it with initiation

Chapter 6 Gene Expression: Translation Translation Nucleotides specify the amino-acid sequence in proteins with four different nucleotides. A, C, G and U. A three letter code (using three of the four nucleotides)

Chapter 10: Protein Synthesis Biology Let s Review What are proteins? Chains of amino acids Some are enzymes Some are structural components of cells and tissues More Review What are ribosomes? Cell structures

Ch. 12: DNA and RNA 12.1 DNA A. To understand genetics, biologists had to learn the chemical makeup of the gene Genes are made of DNA DNA stores and transmits the genetic information from one generation

Transcription & Translation Part of Protein Synthesis Three processes Initiation Transcription Elongation Termination Initiation The RNA polymerase binds to the DNA molecule upstream of the gene at the

Day 1: Transcription Warm Up: What do you think the transcription does? Where does it occur? What is its significance? Lecture: Powerpoint slides with main points bulleted and a few pictures Connection

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide: a closer look A cell translates an message into protein with the help of transfer RNA () Molecules of are not identical: ach carries

Translation Translation: Assembly of polypeptides on a ribosome Living cells devote more energy to the synthesis of proteins than to any other aspect of metabolism. About a third of the dry mass of a cell

Protein Synthesis Protein synthesis is simply the "making of proteins." Although the term itself is easy to understand, the multiple steps that a cell in a plant or animal must go through are not. In order

GENE EXPRESSION AT THE MOLECULAR LEVEL 1 Gene expression Gene function at the level of traits Gene function at the molecular level Two levels tied together since the molecular level affects the structure

Translation Study Guide This study guide is a written version of the material you have seen presented in the replication unit. In translation, the cell uses the genetic information contained in mrna to

Transcription and Translation of DNA Genotype our genetic constitution ( makeup) is determined (controlled) by the sequence of bases in its genes Phenotype determined by the proteins synthesised when genes

Protein Synthesis! From DNA to Protein! (Transcription & Translation)! I. An Overview A. Certain sequences of nucleotides in DNA [called genes], can be expressed/used as a code to determine the sequence

CH 17 Transcription & Translation Basic Principles of Transcription & Translation RNA is the bridge between genes and the proteins for which they code. Transcription is the synthesis of RNA under the direction

DNA Replication & Protein Synthesis This isn t a baaaaaaaddd chapter!!! The Discovery of DNA s Structure Watson and Crick s discovery of DNA s structure was based on almost fifty years of research by other

GENE EXPRESSION Is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as rrna genes

Chapter 14: Gene Transcription and RNA Modification Student Learning Objectives Upon completion of this chapter you should be able to: 1. Understand the role of transcription in gene expression. 2. Know

Chem 121 Chapter 22. Nucleic Acids 1. Any given nucleotide in a nucleic acid contains A) two bases and a sugar. B) one sugar, two bases and one phosphate. C) two sugars and one phosphate. D) one sugar,

Name Period This is going to be a very long journey, but it is crucial to your understanding of biology. Work on this chapter a single concept at a time, and expect to spend at least 6 hours to truly master

Transcription Animations Name: Lew Ports Biology Place http://www.lewport.wnyric.org/jwanamaker/animations/protein%20synthesis%20-%20long.html Protein is the making of proteins from the information found

Tuesday 11/13 Warm Up 1.What are the three parts of a nucleotide? How do two nucleotides link together 2.What binds the two strands of DNA together? Be Specific 3.What are the three main enzymes of DNA

Protein Trafficking/Targeting (8.1) Lecture 8 Protein Trafficking/Targeting Protein targeting is necessary for proteins that are destined to work outside the cytoplasm. Protein targeting is more complex

The process of converting the mrna base sequence into amino acid chains or proteins; occurs in the cytoplasm of the cell on ribosomes The process of converting the mrna base sequence into amino acid chains

Review Questions Translation (protein synthesis) 1. What is the genetic code? The genetic code consists of a sequence of three letter "words" (called 'codons'), written one after another along the length

From DNA to Protein Chapter 14 Impacts, Issues: Ricin and your Ribosomes Ricin is toxic because it inactivates ribosomes, the organelles which assemble amino acids into proteins, critical to life processes

DNA, RNA, Protein Synthesis Keystone 1. During the process shown above, the two strands of one DNA molecule are unwound. Then, DNA polymerases add complementary nucleotides to each strand which results

Protein Synthesis Protein Synthesis How Genes Become Constituent Molecules Mendel and The Idea of Gene What is a Chromosome? A chromosome is a molecule of DNA 50% 50% 1. True 2. False True False Protein

SAM Teacher s Guide DNA to Proteins Note: Answers to activity and homework questions are only included in the Teacher Guides available after registering for the SAM activities, and not in this sample version.

DNA and Genetics 1. Which of the following correctly organizes genetic material from the broadest category to the most specific category? A. genome chromosome gene DNA molecule B. genome chromosome DNA

Peptidyl transferase Uranya: Reynard: If you talk to almost any contemporary molecular biologist or biochemist they will tell you that the answers lie in the RNA World point of view. I know you have already

DNA, RNA, Protein synthesis, and Mutations Chapters 12-13.3 1A)Identify the components of DNA and explain its role in heredity. DNA s Role in heredity: Contains the genetic information of a cell that can

Module 3 Questions Section 1. Essay and Short Answers. Use diagrams wherever possible 1. With the use of a diagram, provide an overview of the general regulation strategies available to a bacterial cell.

Control of Gene Expression (Learning Objectives) Explain the role of gene expression is differentiation of function of cells which leads to the emergence of different tissues, organs, and organ systems

RNA and Protein Synthesis 12.3 Biology Mr. Hines Now we know how DNA (genes) are copied. But how is it used to make a living organism? Most of the structures inside of a cell are made of protein - so we

DNA, RNA AND PROTEIN SYNTHESIS Evolution of Eukaryotic Cells Eukaryotes are larger, more complex cells that contain a nucleus and membrane bound organelles. Oldest eukarytotic fossil is 1800 million years

Study Guide Chapter 12 1. Know ALL of your vocabulary words! 2. Name the following scientists with their contributions to Discovering DNA: a. Strains can be transformed (or changed) into other forms while